Cell signaling II and transcription (Printed).pptx

Cell signalling II Intracellular signalling and the central dogma Dr. Rhea Hurnik BMS 100 Outline: Cell signalling II • 1) Model 1: second messenger activates a protein kinase • cAMP 🡪PKA • Ca2+ 🡪 CaM Kinase • Ras 🡪🡪🡪 Map Kinase • DAG + Ca2+ 🡪 PKC • PI 3 Kinase-AKT pathway • 2) Model 2: Activated...

Cell signalling II Intracellular signalling and the central dogma Dr. Rhea Hurnik BMS 100 Outline: Cell signalling II • 1) Model 1: second messenger activates a protein kinase • cAMP 🡪PKA • Ca2+ 🡪 CaM Kinase • Ras 🡪🡪🡪 Map Kinase • DAG + Ca2+ 🡪 PKC • PI 3 Kinase-AKT pathway • 2) Model 2: Activated cell membrane receptor directly activates a transcription factor • JAK 🡪STAT • Smad • 3) Model 3: Pathways involved in regulated proteolysis • Wnt-Beta catenin pathway • NF𝜅B • 4) Model 4: Intracellular receptors • Steroid hormones • Thyroid hormones • 5) Model 5: non-coding RNA affecting transcription • miRNA • lncRNA Signaling affecting gene transcription The signalling pathways you discussed yesterday were relatively fast, today’s pathways are slower since they involved gene transcription and altered protein synthesis Pathways affecting transcription regulators • Intracellular signalling cascades can affect transcription in a variety of ways: ▪ They can affect transcription factors ▪ They can affect co-activators or co-repressors ▪ They can affect histones remodelling Transcription factor Binds a DNA sequence directly to affect transcription Co-activator/ Co-repressor Binds a transcription factor to affect transcription * Doesn’t binds DNA directly Model 1 Second messenger in a signalling cascade activates a protein kinase involved in transcription regulation + Enzy me 2nd messenger + Protei n kinase Protein kinase TF Modifies gene transcriptio Nucleus Model 1 – shutting it off • Second messenger in a signalling cascade activates a protein kinase involved in transcription regulation + Enzy me 2nd messenger + Protei n kinase Inhibitory enzyme that will shut off the signal + Protein kinase TF Modifies gene transcriptio Nucleus 1) i) cAMP An increase in cytosolic cAMP concentration is stimulated by which cell membrane receptor? - it is stimulated by G-protein coupling receptors cAMP activates cAMP-dependent protein kinase (aka PKA) ▪ Where have we seen this before? 1) i) cAMP activates PKA • PKA is a multimeric protein ▪ Binding of cAMP causes dissociation of catalytic and regulatory subunits Molecular Biology of the Cell (Alberts et al) 6 th ed. Figure 15- 1) i) cAMP activates PKA PKA is a multimeric protein Released catalytic subunits are free to phosphorylate specific target proteins Target proteins can include: • Transcription factors → effects occurs over hours • Adjacent phosphodiesterase ▪ Rapidly lowers cAMP levels to shut off the signal Molecular Biology of the Cell (Alberts et al) 6 th ed. Figure 15- 1) i) cAMP activate PKA to affect transcription To affect transcription regulators: 1. Activated PKA enters the nucleus and activates CREB ▪ CREB = CRE-binding protein • CREB is a transcription factor for genes with a CRE ▪ CRE = cAMP response element ▪ CREB stimulates transcription of genes with a CRE Molecular Biology of the Cell (Alberts et al) 6 th ed. Figure 15- 1) i) cAMP activate PKA to affect transcription • To affect transcription regulators: 2. Activated CREB also recruits a co-activator called CREB-binding protein (CBP) • Further promotes transcription of a gene with a CRE Molecular Biology of the Cell (Alberts et al) 6 th ed. Figure 15- 1) i) Putting it all together • Reminder of the full pathway Molecular Biology of the Cell (Alberts et al) 6 th ed. Figure 15- 1) ii) CaM Kinase • Ca2+/ calmodulin-dependent kinases (CaM-Kinase) can also phosphorylate transcription regulators to increase or decrease transcription ▪ CaM-Kinase can also phosphorylate CREB to increase transcription of genes with a CRE CaMkinase Adapted from Molecular Biology of the Cell (Alberts et al) 6 th ed. Figure 1) ii) Putting it all together • Reminder of the full pathway CaMkinase CaMkinase Adapted from Molecular Biology of the Cell (Alberts et al) 6 th ed. Figure 15-27. Page 836 and Molecular Biology of the Cell (Alberts et al) 6 th ed. 1) iii) PKC • When activated, protein Kinase C (PKC) functions similarly to PKA ▪ It phosphorylates target proteins, which can: • Activate/inhibit proteins in the cell • Activate/inhibit transcription factors OR co-activator/ co-inhibitors to alter gene transcription 1) iii) Putting it all together • Reminder of the full pathway Activate or inhibit transcription factors Activate or inhibit coregulators Adapted from Molecular Biology of the Cell (Alberts et al) 6 th ed. Figure 1 1) iv) Ras • Ras is a protein activated by receptor tyrosine kinases ▪ Active Ras then triggers a series of activation-via-phosphorylation reactions ending with the activation of MAP kinase Molecular Biology of the Cell (Alberts et al) 6 th ed. Figure 1) iv) Ras triggers activation of MAPK • MAP Kinase (Erk) can enter the nucleus to phosphorylate transcription factors ▪ This activates transcription factors needed for immediate early genes • Some of these newly translated proteins then turn on other genes 1) v) PI 3-Kinase AKT pathway A common pathway for growth factors is the PI 3Kinase-AKT pathway AKT activates a wide variety of targets, including • Various transcription factors ▪ Eg. AKT can activate CREB mTOR complex 1 ▪ Activates a variety of targets, including transcription factors involved in ribosome production for increased protein synthesis 1) v) PI 3 Kinase AKT pathway Can activate transcription factors (eg. CREB) AK T *Note: consecutive inhibition (ie. Inhibition of an inhibitory molecule) results in activation Molecular Biology of the Cell (Alberts et al) 6 th ed. Figure 17-64. Model 2 • Activated receptor activates a transcription factor that travels to the nucleus to modify gene transcription + TF TF Modifies gene transcriptio Nucleus Model 2 – shutting it off • Activated receptor activates a transcription factor that travels to the nucleus to modify gene transcription + TF TF Transcription of inhibitory proteins to turn signal off Modifies gene transcriptio Nucleus 2) i) JAK • Janus Kinase (JAK) is a cytosolic tyrosine kinase ▪ JAK is activated by a cytokine ligand binding to its cell membrane receptor • JAK phosphorylates and activates transcription factors called STATS ▪ STAT = signal transducers and activators of transcription • Once activated, STATS travel to the nucleus and regulate gene transcription 2) i) JAK activates STAT Molecular Biology of the Cell (Alberts et al) 6 th ed. Figure 2) ii) Smad Initiated by activation of receptor serine/threonine kinases • Activated by TGF-beta and BMP ligands ▪ TGF- β = Transforming growth factor Beta ▪ BMP = Bone morphogenetic protein • Once activated, the receptor will bind and phosphorylate a transcription factor, Smad ▪ The specific Smad activated depends on the ligand Specific Smad proteins are FYI Molecular Biology of the Cell (Alberts et al) 6th ed. Figure 15-57. Pg 866 2) ii) Smad forms complex • Once Smad is activated, it dissociates from the receptor and forms a complex with a co-Smad ▪ This complex travels to the nucleus and associates with other translation factors and co-regulators to control transcription Specific Smad proteins are FYI (coSmad) Molecular Biology of the Cell (Alberts et al) 6 th ed. Figure Model 3 • Activation of a receptor triggers destruction of an inhibitory protein of a transcription factor. • The transcription factor can the travel to the nucleus to modify gene transcription + T.F. T.F. Modifies gene transcriptio Nucleus 3) Model 3 • Activation of a receptor triggers destruction of an inhibitory protein of a transcription factor. • The transcription factor can the travel to the nucleus to modify gene transcription + Protei n Protei n Transcription of inhibitory proteins to Modifies gene transcriptio Nucleus 3) i) Without Wnt Wnt regulates the proteolysis of a multifunctional protein β-catenin • Without Wnt signalling, β-catenin is phosphorylated and targeted via ubiquitylation for destruction by a β-catenin degradation complex ▪ One protein in this complex is APC ▪ Ubiquitylation targets β-catenin for destruction by proteasome • Wnt-responsive genes are kept silent by an inhibitory complex of transcription regulatory proteins Molecular Biology of the Cell (Alberts et al) 6 th ed. Figure Eg. FYI (for now) Myc **All molecules except Beta-catenin & APC 3) i) Wnt binding frees Betacatenin • Wnt regulates the proteolysis of a multi-functional protein β-catenin ▪ With Wnt signalling, the βcatenin degradation complex is disrupted ▪ Unphosphorylated β-catenin travels to the nucleus ▪ Binding of β-catenin displaces the co-repressor (FYI “Groucho”) and functions as a co-activator Molecular Biology of the Cell (Alberts et al) 6 ed. Figure th Eg. FYI (for now) 3) ii) NF𝜅B • In response to acute inflammatory ligands (IL-1, TNF⍺), a cell surface receptor is activated ▪ Activated receptor triggers the ubiquitylation and phosphorylation to release and destroy an inhibitory protein complex (FYI – I𝜅B) bound to NF𝜅B Molecular Biology of the Cell (Alberts et al) 6th ed. Figure 15-62. Pg 874 **All molecules except NFkB are FYI are FYI 3) ii) NFkB • In response to acute inflammatory ligands (IL-1, TNFalpha), a cell surface receptor is activated ▪ NF𝜅B travels to the nucleus and initiates transcription of NF𝜅B-responsive genes Molecular Biology of the Cell (Alberts et al) 6th ed. Figure 15-62. Pg 874 **All molecules except NFkB are FYI 4) Intracellular receptors • Small, hydrophobic ligands don’t need cell surface receptors since they can easily diffuse across the plasma membrane ▪ Ligands: Steroid hormones, thyroid hormones, retinoids, vitamin D ▪ Their receptors are located inside the cell • Receptors are all structurally similar and are part of a nuclear receptor superfamily **Structures are Molecular Biology of the Cell (Alberts et al) 6 th ed. Figure 15 4) Intracellular receptors continued • Ligand diffuses into the cell and binds to its receptor to alter the ability of the receptor to control transcription of specific genes. ▪ The receptor is BOTH the intracellular receptor AND a transcription factor ▪ A co-regulator is often recruited as well. Model 4 • Hormone ligand binds to an intracellular receptor that, when activated directly modified transcription *Receptor may be in the cytosol or already in the Modifies gene transcriptio Nucleus 4) i) Steroid hormones • Steroid hormone diffuses into the cytoplasm and binds to the receptor in the cytosol ▪ This displaces an inhibitory protein (FYI – hsp) bound to the inactive receptor ▪ Receptor will dimerize and travel to the cell nucleus Image adapted from: Ali Zifan 03:07, 10 July 2016 (UTC), CC BY-SA 4.0 <https://creativecommons.org/licenses/by-sa/4.0>, via 4) i) Steroid hormones • Inside the nucleus the receptor will bind to a DNA sequence specific to the steroid receptor ▪ Hormone response element (HRE) • Coactivator will also bind and transcription will be initiated ▪ (Not shown) Image adapted from: Ali Zifan 03:07, 10 July 2016 (UTC), CC BY-SA 4.0 <https://creativecommons.org/licenses/by-sa/4.0>, via 4) ii) Thyroid hormone • Thyroid hormone receptor is located in the nucleus, already bound to DNA ▪ Binding of thyroid hormone can increase or decrease transcription of genes depending on the gene itself ▪ Thyroid hormone receptors commonly form heterodimers with other nuclear receptors (FYI – RXR = retinoid X receptor) Thyroid Hormone *Note this image is showing the thyroid hormone entering the nucleus from the cytosol! 4) ii) Thyroid hormone Thyroid hormone receptor is located in the nucleus, already bound to DNA ▪ Binding of thyroid hormone can increase or decrease transcription of genes depending on the gene itself • Positively regulated genes will have increased transcription when thyroid hormone binds to its receptor • Negatively regulated genes will have decreased transcription when thyroid hormone binds to its receptor 5) Modification by noncoding RNA Gene expression can also be regulated by noncoding RNAs: ▪ miRNA • Mature miRNA is ~21-30 nucleotides in length (FYI) • Modulate translation of target messenger RNAs (mRNAs) • Post-transcriptional silencing of gene expression by miRNA is a fundamental mechanism of gene regulation present in all eukaryotes • Each miRNA can modulate activity of multiple protein-coding genes ▪ lncRNA • >200 nucleotides in length (FYI) • Can bind chromatin to interfere or promote transcription • Also involved in X chromosome inactivation 4)i) miRNA • miRNA process: ▪ Transcription of miRNA forms a primary transcript (pri-miRNA) ▪ Further processing by a number of enzymes (eg. Dicer) produces smaller and smaller miRNA segments • Active product is called miRNA 4) i) miRNA cont. miRNA process: • miRNA associates with proteins to form a RNA-induced silencing complex (RISC) • Base-pairing of the miRNA (within RISC) can either: ▪ Induce mRNA cleavage/destruction ▪ Repress translation The net effect is that miRNAs within a RISC complex act to silence mRNA post-transcription 4) ii) lncRNA lncRNA can function is many ways to modify transcription by: ▪ A) promote gene transcription ▪ B) suppress gene transcription ▪ C) Promote chromatin modification directly ▪ D) Stabilize protein complexes that modify chromatin structure Team activity time Colorectal cancer is the 4th most commonly diagnosed cancer in Canada. ▪ The tumor itself (colorectal adenocarcinoma) tends to grow slowly and can get quite large before symptoms will present. ▪ Initial presenting symptoms can include anemia with fatigue and weakness, bleeding with defecation, or bowel obstruction. • 70-80% of nonfamilial colorectal cancers involve defects in genes for APC within the Wnt/β-catenin pathway, resulting in APC loss of function. APC loss of function has been implication is the pathogenesis of colonic tumours. • Myc is a Wnt-responsive gene. ▪ Myc is a gene that codes for the transcription factor MYC, which has been shown to drive cell proliferation. Team activity time Questions • 1. Draw out the basic schematic of the Wnt-β catenin pathway ▪ Which model did it belong to? • 2. In 2-3 bullet points clearly explain what would happen to gene transcription for Wnt-responsive genes. • 3. In 2-3 bullet points connect the loss of APC to development of colon cancer. In your answer, please connect the role of Myc. References • Boron, W. and Boulpaep, E. Medical Physiology (3rd ed). Elsevier • Alberts et al. Molecular Biology of the Cell. Garland Science. • Robbins and Cotran. Pathologic Basis of Disease. Elsevier • Images: ▪ Image adapted from: Boghog2, Public domain, via Wikimedia Commons. Retrieved from: https://commons.wikimedia.org/wiki/File:Type_ii_nuclear_receptor_action.png ▪ Image adapted from: Ali Zifan 03:07, 10 July 2016 (UTC), CC BY-SA 4.0 <https://creativecommons.org/licenses/by-sa/4.0>, via Wikimedia Commons. Retrived from: https://commons.wikimedia.org/wiki/File:Regulation_of_gene_expression_by_st eroid_hormone_receptor.svg